Chronic pneumonia with Pseudomonas aeruginosa and impaired alveolar fluid clearance

Background

While the functional consequences of acute pulmonary infections are widely documented, few studies focused on chronic pneumonia. We evaluated the consequences of chronic Pseudomonas lung infection on alveolar function.

Methods

P. aeruginosa, included in agar beads, was instilled intratracheally in Sprague Dawley rats. Analysis was performed from day 2 to 21, a control group received only sterile agar beads. Alveolar-capillary barrier permeability, lung liquid clearance (LLC) and distal alveolar fluid clearance (DAFC) were measured using a vascular (¹³¹I-Albumin) and an alveolar tracer (¹²⁵I-Albumin).

Results

The increase in permeability and LLC peaked on the second day, to return to baseline on the fifth. DAFC increased independently of TNF-α or endogenous catecholamine production. Despite the persistence of the pathogen within the alveoli, DAFC returned to baseline on the 5^th day. Stimulation with terbutaline failed to increase DAFC. Eradication of the pathogen with ceftazidime did not restore DAFC response.

Conclusions

From these results, we observe an adequate initial alveolar response to increased permeability with an increase of DAFC. However, DAFC increase does not persist after the 5^th day and remains unresponsive to stimulation. This impairment of DAFC may partly explain the higher susceptibility of chronically infected patients to subsequent lung injury.     

Malnutrition impairs alveolar fluid clearance in rat lungs

Inadequate nutrition complicates the clinical course of critically ill patients, and many of these patients develop pulmonary edema. However, little is known about the effect of malnutrition on the mechanisms that resolve alveolar edema. Therefore, we studied the mechanisms responsible for the decrease in alveolar fluid clearance in rats exposed to malnutrition. Rats were allowed access to water, but not to food, for 120 h. Then, the left and right lungs were isolated for the measurement of lung water volume and alveolar fluid clearance, respectively. The rate of alveolar fluid clearance was measured by the progressive increase in the concentration of Evans blue dye that was instilled into the distal air spaces with an isosmolar 5% albumin solution over 1 h. Malnutrition decreased alveolar fluid clearance by 38% compared with controls. Amiloride (10(-3) M) abolished alveolar fluid clearance in malnourished rats. Either refeeding for 120 h following nutritional deprivation for 120 h or an oral supply of sodium glutamate during nutritional deprivation for 120 h restored alveolar fluid clearance to 91 and 86% of normal, respectively. Dibutyryl-cGMP, a cyclic nucleotide-gated cation channel agonist, increased alveolar fluid clearance in malnourished rats supplied with sodium glutamate. Terbutaline, a beta(2)-adrenergic agonist, increased alveolar fluid clearance in rats under all conditions (control, malnutrition, refeeding, and glutamate-treated). These results indicate that malnutrition impairs primarily amiloride-insensitive and dibutyryl-cGMP-sensitive alveolar fluid clearance, but this effect is partially reversible by refeeding, treatment with sodium glutamate, or beta-adrenergic agonist therapy.     

CD1d-dependent macrophage-mediated clearance of Pseudomonas aeruginosa from lung

CD1d-restricted T cells are implicated as key players in host defense against various microbial infections. However, the mechanisms involved and the role they play, if any, at the mucosal surfaces where pathogenic infections are initiated is unknown. In a murine pneumonia model established by intranasal application of Pseudomonas aeruginosa, CD1d(-/-) mice showed markedly reduced pulmonary eradication of P. aeruginosa compared with wild-type mice; this was associated with significantly lower amounts of macrophage inflammatory protein-2 and reduced numbers of neutrophils within the bronchoalveolar lavage fluid. Corollarily, treatment of mice with alpha-galactosylceramide--a lipid that activates CD1d-restricted T cells--increased the amount of interferon-gamma; this was associated with rapid pulmonary clearance through enhanced phagocytosis of P. aeruginosa by alveolar macrophages. These results reveal a crucial role played by CD1d-restricted T cells in regulating the antimicrobial immune functions of macrophages at the lung mucosal surface.     

Immunisation with non-integral OMPs promotes pulmonary clearance of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause fatal acute lung infections in critically ill individuals. Lung damage due to chronic infections in cystic fibrosis sufferers is the major cause of morbidity and mortality in this group. The bacterium produces various immunomodulatory products that enable it to survive in the lung. Innate and increasing resistance to antibiotic therapy shown by this organism heightens the need for development of a vaccine. This study reports the identification of six non-integral protein antigens; Pa13, azurin, acyl carrier protein (ACP), amidase, aminopeptidase and KatE, purified from a mucoid strain of P. aeruginosa. N-terminal amino acid sequencing was used to identify these proteins and, based on their ascribed functions, determined that their normal cellular location was cytosolic. A rat model of acute pulmonary infection was used to investigate the ability of these protein antigens to enhance pulmonary clearance of a live P. aeruginosa challenge. Mucosal immunisation with four of the six antigens significantly enhanced bacterial clearance from both the lavage fluid and lung tissue. The greatest level of clearance was demonstrated for the antigens; KatE, aminopeptidase and amidase. Enhanced bacterial clearance was maintained when the antigens amidase and aminopeptidase were produced in recombinant form. When delivered parenterally, aminopeptidase demonstrated its continued efficacy as a vaccine candidate. This study has demonstrated that non-integral outer membrane proteins are antigenic and protective and warrant further investigation as potential components of a vaccine.     

Substrate interaction with recombinant amidase from Pseudomonas aeruginosa during biocatalysis

The interaction of a variety of substrates with Pseudomonas aeruginosa native amidase (E.C. 3.5.1.4), overproduced in an Escherichia coli strain, was investigated using difference FTIR spectroscopy. The amides used as substrates showed an increase in hydrogen bonding upon association in multimers, which was not seen with esters. Evidence for an overall reduction or weakening of hydrogen bonding while amide and ester substrates are interacting with the enzyme is presented. The results describe a spectroscopic approach for analysis of substrate–amidase interaction and in situ monitoring of the hydrolysis and transferase reaction when amides or esters are used as substrates.     

Pretreatment with the Gram-positive bacterial cell wall molecule peptidoglycan improves bacterial clearance and decreases inflammation and mortality in mice challenged with Pseudomonas aeruginosa

The objective of this study was to determine if inflammatory tolerance and enhancement of innate immune function could be induced by the Gram-positive cell wall component peptidoglycan (PGN). Male mice (C57BL6/J or C3H/HeJ, 8-12 weeks of age) were given intraperitoneal injections of 1mg PGN on 2 consecutive days. The mice were then challenged with lipopolysaccharide (LPS) or live Pseudomonas aeruginosa (1 x 10(8) colony-forming units) 2 days after the second pretreatment. Mice pretreated with PGN had diminished plasma concentrations of TNFalpha and IFNgamma and elevated concentrations of IL-10 in response to a subsequent LPS or Pseudomonas challenge when compared to untreated controls. Bacterial clearance was improved in mice pretreated with PGN, and mortality in response to a subsequent Pseudomonas challenge was significantly attenuated. PGN pretreatment of LPS-unresponsive mice (C3H/HeJ) verified that the effect of PGN pretreatment was not due to any LPS contamination. We have previously demonstrated that PGN pretreatment induced resistance to a Gram-positive bacterial challenge. The present study extends those results by showing that exposure to the Gram-positive bacterial cell wall component peptidoglycan also induces cross-tolerance to LPS and non-specifically enhances innate immune function in that PGN-pretreated mice had increased resistance to Gram-negative bacterial challenge.     

Enzymatic Degradation of Ribosomes During Endogenous Respiration of Pseudomonas aeruginosa.

Gronlund, Audrey F. (University of British Columbia, Vancouver, B.C., Canada), and J. J. R. Campbell. Enzymatic degradation of ribosomes during endogenous respiration of Pseudomonas aeruginosa. J. Bacteriol. 90:1-7. 1965.-From sedimentation analyses it was found that the ribosomal content of Pseudomonas aeruginosa decreased during endogenous respiration. A greater degree of degradation of 50S than 30S ribosomes occurred during the 3-hr starvation period. The enzyme responsible for the initiation of ribosome degradation and present in the ribosome fraction was identified as polynucleotide phosphorylase. The enzyme was inactive in intact 70S ribosomes, but was active in low magnesium ion concentrations which allowed the 70S ribosome to dissociate. Polynucleotide phosphorylase was not solubilized after dissociation of the 70S particle, but remained firmly attached to the 50S and 30S ribosomes, the ribonucleic acid of which served as substrate.     

Isolation of Pseudomonas aeruginosa and other bacterial species from ornamental aquarium plants.

Ornamental aquarium plants were demonstrated to carry as many as 10(8) viable mesophilic bacteria per g. Gram-negative organisms predominated among the 19 genera of bacteria identified. Pseudomonas aeruginosa was readily isolated from 53% of the samples tested.     

Production and purification of the bacterial autolysin N-acetylmuramoyl-L-alanine amidase B from Pseudomonas aeruginosa

The bacterial cell wall heteropolymer peptidoglycan is not a static structure as it is constantly being made and recycled throughout the bacterium's life cycle. This turnover of peptidoglycan is a highly coordinated event involving a complement of autolytic enzymes that include those with specificity for either the carbohydrate or the peptide linkages of peptidoglycan. One major class of these autolysins are the N-acetylmuramoyl-L-alanine amidases which cleave the amide linkage between the stem peptides and the lactyl moiety of muramoyl residues. They are required in the periplasm for cell separation during division and in both the periplasm and cytoplasm to trim soluble released PG fragments during turnover for recycling. The gene encoding N-acetylmuramoyl-L-alanine amidase B in Pseudomonas aeruginosa was cloned and over-expressed in Escherichia coli. The recombinant protein with a C-terminal His-tag was purified to apparent homogeneity by a combination of affinity and cation-exchange chromatographies using Ni(2+)NTA-agarose and Source S, respectively. Four separate assays involving zymography, light scattering, HPLC and MALDI-TOF mass spectrometry were used to confirm the activity of the protein as an N-acetylmuramoyl-L-alanine amidase.     

Cardiolipin-mediated PPARγ S112 phosphorylation impairs IL-10 production and inflammation resolution during bacterial pneumonia

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Transgenic cystic fibrosis mice exhibit reduced early clearance of Pseudomonas aeruginosa from the respiratory tract

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) has been proposed to be an epithelial cell receptor for Pseudomonas aeruginosa involved in bacterial internalization and clearance from the lung. We evaluated the role of CFTR in clearing P. aeruginosa from the respiratory tract using transgenic CF mice that carried either the DeltaF508 Cftr allele or an allele with a Cftr stop codon (S489X). Intranasal application achieved P. aeruginosa lung infection in inbred C57BL/6 DeltaF508 Cftr mice, whereas DeltaF508 Cftr and S489X Cftr outbred mice required tracheal application of the inoculum to establish lung infection. CF mice showed significantly less ingestion of LPS-smooth P. aeruginosa by lung cells and significantly greater bacterial lung burdens 4.5 h postinfection than C57BL/6 wild-type mice. Microscopy of infected mouse and rhesus monkey tracheas clearly demonstrated ingestion of P. aeruginosa by epithelial cells in wild-type animals, mostly around injured areas of the epithelium. Desquamating cells loaded with P. aeruginosa could also be seen in these tissues. No difference was found between CF and wild-type mice challenged with an LPS-rough mucoid isolate of P. aeruginosa lacking the CFTR ligand. Thus, transgenic CF mice exhibit decreased clearance of P. aeruginosa and increased bacterial burdens in the lung, substantiating a key role for CFTR-mediated bacterial ingestion in lung clearance of P. aeruginosa.     

Iron reductases from Pseudomonas aeruginosa

Cell-free extracts of Pseudomonas aeruginosa contain enzyme activities which reduce Fe(III) to Fe(II) when iron is provided in certain chelates, but not when the iron is uncomplexed. Iron reductase activities for two substrates, ferripyochelin and ferric citrate, appear to be separate enzymes because of differences in heat stabilities, in locations in fractions of cell-free extracts, in reductant specificity, and in apparent sizes during gel filtration chromatography. Ferric citrate iron reductase is an extremely labile activity found in the cytoplasmic fraction, and ferripyochelin iron reductase is a more stable activity found in the periplasmic as well as cytoplasmic fraction of extracts. A small amount of activity detectable in the membrane fraction seemed to be loosely associated with the membranes. Although both enzymes have highest activity reduced nicotinamide adenine dinucleotide, reduced glutathione also worked with ferripyochelin iron reductase. In addition, oxygen caused an irreversible loss of a percentage of the ferripyochelin iron reductase following sparge of reaction mixtures, whereas the reductase for ferric citrate was not appreciably affected by oxygen.     

The Pseudomonas aeruginosa proteome during anaerobic growth

Isotope-coded affinity tag analysis and two-dimensional gel electrophoresis followed by tandem mass spectrometry were used to identify Pseudomonas aeruginosa proteins expressed during anaerobic growth. Out of the 617 proteins identified, 158 were changed in abundance during anaerobic growth compared to during aerobic growth, including proteins whose increased expression was expected based on their role in anaerobic metabolism. These results form the basis for future analyses of alterations in bacterial protein content during growth in various environments, including the cystic fibrosis airway.     

Structural and functional basis of phospholipid oxygenase activity of bacterial lipoxygenase from Pseudomonas aeruginosa

Pseudomonas aeruginosa expresses a secreted LOX-isoform (PA-LOX, LoxA) capable of oxidizing polyenoic fatty acids to hydroperoxy derivatives. Here we report high-level expression of this enzyme in E. coli and its structural and functional characterization. Recombinant PA-LOX oxygenates polyenoic fatty acids including eicosapentaenoic acid and docosahexaenoic acid to the corresponding (n-6)S-hydroperoxy derivatives. This reaction involves abstraction of the proS-hydrogen from the n-8 bisallylic methylene. PA-LOX lacks major leukotriene synthase activity but converts 5S-HETE and 5S,6R/S-DiHETE to anti-inflammatory and pro-resolving lipoxins. It also exhibits phospholipid oxygenase activity as indicated by the formation of a specific pattern of oxygenation products from different phospholipid subspecies. Multiple mutagenesis studies revealed that PA-LOX does not follow classical concepts explaining the reaction specificity of mammalian LOXs. The crystal structure of PA-LOX was solved with resolutions of up to 1.48 Å and its polypeptide chain is folded as single domain. The substrate-binding pocket consists of two fatty acid binding subcavities and lobby. Subcavity-1 contains the catalytic non-heme iron. A phosphatidylethanolamine molecule occupies the substrate-binding pocket and its sn1 fatty acid is located close to the catalytic non-heme iron. His377, His382, His555, Asn559 and the C-terminal Ile685 function as direct iron ligands and a water molecule (hydroxyl) completes the octahedral ligand sphere. Although the biological relevance of PA-LOX is still unknown its functional characteristics (lipoxin synthase activity) implicate this enzyme in a bacterial evasion strategy aimed at downregulating the hosts' immune system.     

Modeling and simulations of a bacterial outer membrane protein: OprF from Pseudomonas aeruginosa

OprF is a major outer membrane protein from Pseudomonas aeruginosa, a homolog of OmpA from Escherichia coli. The N-terminal domains of both proteins have been demonstrated to form low conductance channels in lipid bilayers. Homology models, consisting of an eight-stranded beta-barrel, of the N-terminal domain OprF have been constructed based on the crystal structure of the corresponding domain from E. coli OmpA. OprF homology models have been evaluated via a set (6 x 10 ns) of simulations of the beta-barrel embedded within a solvated dimyristoyl-phosphatidylcholine (DMPC) bilayer. The conformational stability of the models is similar to that of the crystal structure of OmpA in comparable simulations. There is a degree of water penetration into the pore-like center of the OprF barrel. The presence of an acidic/basic (E8/K121) side-chain interaction within the OprF barrel may form a "gate" able to close/open a central pore. Lipid-protein interactions within the simulations were analyzed and revealed that aromatic side-chains (Trp, Tyr) of OprF interact with lipid headgroups. Overall, the behavior of the OprF model in simulations supports the suggestion that this molecule is comparable to OmpA. The simulations help to explain the mechanism of formation of low conductance pores within the outer membrane.